Traditionally, hematoxylin and eosin staining (H&E) is one of the most common staining methods in histology. The morphology from the H&E is most widely used in medical diagnosis for cancer detection. However, increasingly molecular analysis of tissue by immunohistochemistry (IHC) or immunofluorescence (IF) and fluorescence in situ hybridization have become an essential part of cancer diagnosis in addition to morphological assessment. With great strides in targeted therapies, detailed molecular assessment of cancer tissue is fast becoming a requirement. Additionally with early cancer detection sample size is diminishing, making it difficult to perform various analyses required for complete characterization of disease.
While valuable, many of these current techniques may detect only a few targets at one time (such as IHC or fluorescence-based Western blots where number of targets detectable is limited by the fluorescence-based detection system) in a single sample. Further analysis of targets may require use of additional biological samples from the source, limiting the ability to determine relative characteristics of the targets such as the presence, absence, concentration, and/or the spatial distribution of multiple biological targets in the biological sample. Moreover, in certain instances, a limited amount of sample may be available for analysis or the individual sample may require further analysis.
Furthermore in many older cases only samples available are H&E stained slides and as patients are relapsing molecular analysis of this tissue can significantly benefit these patients by matching their disease to available targeted therapies. A major problem with using the previously H&E stained slide for IHC and FISH is the interference from H&E stains in both chromogenic detection in IHC and fluorescence detection for IF and FISH. Attempts have been made to remove H&E from tissue for interrogation. While this has allowed IHC with chromogen detection, IF and FISH are not feasible due to strong residual fluorescence from eosin. Removal of residual eosin fluorescence has not been feasible with many different techniques attempted to date.
For this reason, the general practice has been to use different tissue sections for H&E and molecular analysis. In rare cases where previously stained H&E slides are the only sample available, partial removal of H&E is performed and the slides are used for IHC using a chromogenic signal (Benharroch et. al. Blood 1998, 91:2076-2084). In limited circumstances it may also be possible to perform IF or FISH using a fluorophore whose emission is far removed from the eosin fluorescence, however, most of the commercial probes or labeled antibodies are labeled with fluorphores whose emission overlaps with eosin emission.
Thus there still remains a need for a method to remove H &E signals from the tissue and use the same tissue section for subsequent analysis such as immunofluorescence and FISH.